This page contains detailed protocols for the experiments in ‘Colour biases in learned foraging preferences in Trinidadian guppies’. We provide step by step instructions on how to perform the training, testing, and behavioural quantification described in the manuscript. If you would like to see details on housing and husbandry conditions during the experiment please refer to section 2 of the manuscript.
For this project, we wanted to determine whether object preferences were plastic for both green and blue objects in Trinidadian guppies. Given these colours differ in their foraging relevancy for guppies we wanted to determine whether the shift in preference would be unconstrained. Our protocol aimed to manipulate object preferences for the green and blue objects (Figure 2) of individual guppies by manipulating their foraging experiences with these objects. This was done by providing food reinforcement for approaching one of the two colours.
On the first trial, guppies were placed in the test tank with two objects, one blue and one green, but with empty food strips to measure initial preferences. Training then occurred once daily between 10h00 to 16h00 for 20 days constituting 20 trials. During training, the location of the rewarding object (left or right) was randomized across days and individuals. That is, only the object’s appearance provided a reliable cue to food location. On the 21st day, an unreinforced test was given where new duplicates of the objects were presented with empty plastic strips attached to them. In both experiments this unreinforced trial (Trial 21, Probe 1) was used to assess whether learning had occurred. At this point, experiment 1 ended. In experiment 2 an additional four test trials were conducted to see how robust learning was in both groups across different contexts.
Figure 1: An experimental timeline representing the order of the trials. Experiment 1 ends at trial 21. Experiment 2 ends at trial 29.
In the video below we provide sample footage of the behavioural assays conducted during the experiments. The footage below is of a green-trained guppy throughout experiment 2 and demonstrates the types of trials guppies were exposed to in this project (see Figure 2 for details on trials).
For the initial test and final tests guppies were presented with the green and blue objects with empty food strips attached to them. You can see an example of this above. The following protocol was used for testing:
Guppies were rewarded for foraging from either the blue object or the green object, forming the two experimental treatments, blue-rewarded and green-rewarded. The food reward was a gelatin mixture of flake food which was placed on the back of the objects facing away from the guppy so that the rewarding object had to be approached and investigated for the food reward to be discovered. To see how to make the food reward go to Food strips. The protocol was as follows:
The food strips that were attached to the objects were made with the following protocol:
Figure 2: (A) The two objects used in experiment 1. Blue-trained guppies were trained to the left object and green-trained guppies were trained to the right object. The manufacturer’s colour name for the blue object is ‘dark azur (hex #078BC9) and for the green object is ‘bright yellowish green’ (hex # BBE90B). (B) The 7 pairs of objects in experiment 2. Object pairs 1,2,3,4, and A had the same colour properties as the object pair used in experiment 1. The bricks for the generalization objects were ‘dark green’ (hex #237841) and ‘bright blue’ (#0055BF) as specified by the manufacturer. The numbered object pairs are used for training while the lettered object pairs are used for testing. All Lego objects were placed in the tank with the studs facing up except object pair 1 which was placed in the tank with studs facing to the side as in the image. The experimental timeline represents the order of the trials. Experiment 1 ends at trial 21. Experiment 2 ends at trial 29.
The colours used in the experiment are listed in Table 1.
| Object Colour | Colour Family | Exact Colour | Hex Value |
|---|---|---|---|
| Green | Dark Green | Bright Yellowish Green | BBE90B |
| Green | Dark Green | Dark Green | 237841 |
| Blue | Blue | Dark Azur | 078BC9 |
| Blue | Blue | Bright blue | 0055BF |
The objects were 6 pairs of Lego blocks with the manufacturer colours A and B. The specific models for the object pairs are listed in Table 2. As of 2021, these bricks can be looked up and/or purchased using the official Lego store’s Choose a brick service by entering the design ID in the Enter an item number or design ID prompt of the Enter set number section.
| Object Pair | Brick Style | Design ID |
|---|---|---|
| Pair A | BRICK 1X2 | 3004 |
| Pair B | BRICK 2x4 | 3001 |
| Pair 1 | BRICK 1X2 (x2) | 3004 |
| Pair 2 | BRICK 2X4 | 3001 |
| Pair 3 & Exp. 1 Green object | BRICK 2X2 | 30165 |
| Pair 4 | BRICK 1X1 | 3005 |
| Exp. 1 Blue object | BRICK 1X3 | 3622 |
We quantified behaviour throughout this project using EthoVision XT 11.5. Below are the instructions that were given to an undergraduate student (Mina Araya-Yohannes) tasked with conducting experimental trials, and collecting as well as quantifying behavioural data for a large portion of experiment 2 trials. While EthoVision is proprietary software, we reproduce our protocol for using it in this experiment for those who may have it or intend to use it.
L://MP_ROOT/.mp4 video files from the trials of the current day into a
folder named after the trial number (e.g., trial-x/) found within the
colour-learning-experiment-2/videos/ folder..mp4 files based on the IDs of the fish. Each video file should
be named after a specific fish.New from templateUse a custom templateBrowse... and navigate to
colour-learning-experiment-2/trial-1/trial-1.evxt to use the experiment
template from this fileOKBrowse... and navigate to colour-learning-experiment-2/ and name
the file trial-x where x is the number of the trial for that day (this can
be found on the data sheet).Arena Settings tab and select Arena Settings 1Background.Open.Grab and make sure the arena graphics still line up then click
Validate Setup.Trial ListAdd Trials in the top left corner and add 16 trialsAcquisition (0 acquired)Playback Control to set the
background image.... next to Video under the Settings tab to the rightAnalysis tab go to the Results tab and click Statistics & ChartsCalculateExport tab click StatisticsOK, the default settings should be sufficientcolour-learning-experiment-2/trial-x/Export Files/experiment-2-raw-data/Statistics-trial-x into the Google drive folder.